The studies described here are concerned with elucidating the molecular and physiological basis of rhodopsin regeneration. Specifically, the studies are focused on understanding the process by which 11-cis-retinol, the ultimate precursor of the visual chromophore, is synthesized in the vertebrate pigment epithelium. We have shown that the membranes of the pigment epithelium contain a novel biosynthetic enzyme system capable of transforming free all-trans-retinol into its 11-cis congener. This thermodynamically uphill process derives its energy from the phospholipids of the membranes in a novel process which utilizes the free energy of hydrolysis of the ester bonds to drive the isomerization. The mechanism employed involves the transesterification of all-trans-retinol with a phospholipid followed by enzymatic processing of the retinyl ester directly to 11-cis-retinol. We have solubilized and partially purified the enzymes involved. In this grant request, we propose to completely purify the enzyme components of this isomerization process, namely, the retinyl ester synthetase and isomerase. We propose to study the mechanisms of action of these enzymes and prepare mechanism-based enzyme inhibitors of them to be used to study their physiological roles. Furthermore, monoclonal and monospecific polyclonal antibodies will be prepared against the enzymes. These antibodies will be used to determine the steady state levels, the turnover rates, and the possible post-translational modification of the enzymes. The mechanisms by which the enzymes might be regulated as well as the mechanism by which the enzymes interact with the membrane are of exceptional interest here. The availability of sequence information on the enzymes and the availability of specific antibodies to the enzymes should enable us to clone them and fully sequence them. This information is important for determining what these enzymes are related to, what their major structural motifs might be, and how the enzymes interact with the membrane. Sequence information will also be of interest clinically for determining whether or not these enzymes, which are the sine qua non of vertebrate vision, are altered in major diseases of vision.